Post-meiotic DNA double-strand breaks are conserved in fission yeast.

In mammals, spermiogenesis is characterized by transient formation of DNA double-strand breaks (DSBs) in the whole population of haploid spermatids. DSB repair in such haploid context may represent a mutational transition. Using a combination of pulsed-field gel electrophoresis and specific labelling of DSBs at 3'OH DNA ends, we showed that post-meiotic, enzyme-induced DSBs are also observed in the synchronizable pat1-114 mutant of Shizosaccharomyces pombe as well as in a wild-type strain, while DNA repair is observed at later stages. This transient DNA fragmentation arises in the whole cell population and is seemingly independent of the caspase apoptotic pathway. Because histones are still present in spores, the transient DSBs do not require a major change in chromatin structure. These observations confirm the highly-conserved nature of the process in eukaryotes and provide a powerful model to study the underlying mechanism and its impact on the genetic landscape and adaptation.

Quantification and genome-wide mapping of DNA double-strand breaks.

DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3'-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair.

Step-specific Sorting of Mouse Spermatids by Flow Cytometry.

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

Instability of trinucleotidic repeats during chromatin remodeling in spermatids.

Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some trinucleotidic repeats diseases.

Male-driven de novo mutations in haploid germ cells.

At the sequence level, genetic diversity is provided by de novo transmittable mutations that may act as a substrate for natural selection. The gametogenesis process itself is considered more likely to induce endogenous mutations and a clear male bias has been demonstrated from recent next-generation sequencing analyses. As new experimental evidence accumulates, the post-meiotic events of the male gametogenesis (spermiogenesis) appear as an ideal context to induce de novo genetic polymorphism transmittable to the next generation. It may prove to be a major component of the observed male mutation bias. As spermatids undergo chromatin remodeling, transient endogenous DNA double-stranded breaks are produced and trigger a DNA damage response. In these haploid cells, one would expect that the non-templated, DNA end-joining repair processes may generate a repertoire of sequence alterations in every sperm cell potentially transmittable to the next generation. This may therefore represent a novel physiological mechanism contributing to genetic diversity and evolution.

"Breaking news" from spermatids.

During the haploid phase of spermatogenesis, spermatids undergo a complex remodeling of the paternal genome involving the finely orchestrated replacement of histones by the highly-basic protamines. The associated striking change in DNA topology is characterized by a transient surge of both single- and double-stranded DNA breaks in the whole population of spermatids which are repaired before spermiation. These transient DNA breaks are now considered part of the normal differentiation program of these cells. Despite an increasing interest in the study of spermiogenesis in the last decade and the potential threat to the haploid genome, the origin of these DNA breaks still remains elusive. This review briefly outlines the current hypotheses regarding possible mechanisms that may lead to such transient DNA fragmentation including torsional stress, enzyme-induced breaks, apoptosis-like processes or oxidative stress. A better understanding of the origin of these DNA breaks will lead to further investigations on the genetic instability and mutagenic potential induced by the chromatin remodeling.

Lors de la phase haploïde de la spermatogenèse, les spermatides subissent un remodelage complexe du génome paternel impliquant un remplacement finement orchestré des histones par des protamines hautement basiques. Le changement topologique de l'ADN associé est caractérisé par une augmentation transitoire de cassures simple et double brins de l'ADN dans l'entière population des spermatides qui sont réparées avant la spermiation. Ces cassures transitoires de l'ADN sont maintenant considérées comme faisant partie du processus normal de différenciation de ces cellules. Malgré un intérêt croissant dans l'étude de la spermiogenèse ces 10 dernières années et la menace potentielle pour le génome haploïde, l'origine de ces cassures d'ADN reste encore incertaine. Cette revue décrit brièvement les hypothèses actuelles concernant les mécanismes possibles qui pourraient mener à cette fragmentation transitoire de l'ADN incluant le stress torsionnel, les cassures enzymatiques, des processus semblables à l'apoptose et le stress oxidatif. Une meilleure compréhension de l'origine de ces cassures d'ADN mènerait à des études approfondies concernant l'instabilité génétique et le potentiel mutagène induit par le remodelage de la chromatine.